The invention relates to genetic modification of plants, plant cells and seeds, particularly altering fatty acid composition.
Soybean (Glycine max) is one of the highest value crops currently grown in the United States (≈$16 billion in 1996). Ranking close to corn (25%) and wheat (22%), soybean accounted for 19% of the United States crop acres planted in 1994. Often referred to as a xe2x80x9cmiracle cropxe2x80x9d, soybean offers tremendous value through the oil, protein and whole soybean products. Agronomic traits, food quality traits related to oils and protein quality are all important for the soybean industry.
More soybeans are grown in the United States than anywhere else in the world (2.4 billion bushels in 1996, 50% of world production). A bushel of soybean (60 pounds) is comprised of 48 pounds of protein meal and 11 pounds of oil. While protein meal is the major component in soybean, oil, lecithin, tocopherols, isoflavones, etc. are all co-products and add value to the bean. Soybean oil is the major edible oil used in the world (40% of the 59.4 million metric tons in 1993). It also accounts for 70% of the 14 billion pounds of edible vegetable oil in the United States. The primary food applications where the oil is used extensively are for baking and frying (40-45%), salad and cooking oil (40-45%), margarine and shortening (15-20%) and a wide spectrum of processed foods. Development of other vegetable oils for specialty uses has recently affected the acreage and production of soybean. The low cost and ready availability of soybean oil provide an excellent opportunity to upgrade this commodity item for specialty uses.
Food fats and oils are chemically composed of triesters of glycerol containing straight chain, normal aliphatic fatty acids, also referred to herein as triacylglycerols or triglycerides (TAG). The properties of food fats and oils are a reflection of the fatty acids contained in the TAG and their distribution on the glycerol backbone. When the melting point of the TAG is below room temperature, the TAG is referred to as an xe2x80x9coilxe2x80x9d. Triglycerides that melt above room temperature are referred to as xe2x80x9cfatxe2x80x9d. Gradients between fluidity and solidity exist. Partially solidified, non-pourable triglycerides are often referred to as xe2x80x9cplastic fatsxe2x80x9d.
Fatty acids are organic acids having a hydrocarbon chain ranging in length from about 4 to 24 carbons. Fatty acids differ from each other in chain length, and in the presence, number and position of double bonds. In cells, fatty acids typically exist in covalently bound forms, the carboxyl portion being referred to as a fatty acyl group. The chain length and degree of saturation of these molecules is often depicted by the formula CX:Y, where xe2x80x9cXxe2x80x9d indicates number of carbons and xe2x80x9cYxe2x80x9d indicates number of double bonds.
Typically, oil derived from commercial soybean varieties is composed of approximately 11% palmitic (C16:0), 4% stearic acid (C18:0), 21% oleic acid (C18:1), 56% linoleic acid (C18:2), and 10% linolenic acid (C18:3). The fatty acid composition of soybean oil, as well as all oils, largely determines its physical and chemical properties, and thus its uses.
Fatty acid biosynthesis has been the subject of research efforts in a number of organisms. For reviews of fatty acid biosynthesis in plants, see Ohlrogge et al., (1995) Plant Cell, 7:957-970, Ohlrogge et al., (1997) Annu Rev Plant Physiol Plant Mol Biol, 48:109-136 and Sommerville et al. (1991) Science, 252:80-87.
As mentioned previously, the fatty acid composition of an oil determines its physical and chemical properties, and thus its uses. Plants, especially plant species which synthesize large amounts of oils in plant seeds, for example soybean, are can important source of oils both for edible and industrial uses. Various combinations of fatty acids in the different positions in the triglyceride will alter the properties of the triglyceride. For example, if the fatty acyl groups are mostly saturated fatty acids, then the triglyceride will be solid at room temperature. In general, however, vegetable oils tend to be mixtures of different triglycerides. The triglyceride oil properties are therefore a result of the combination of triglycerides which make up the oil, which are in turn influenced by their respective fatty acid compositions.
Plant breeders have successfully modified the yield and fatty acid composition of various plant seed oils by introducing desired traits through plant crosses and selection of progeny carrying the desired trait forward. Application of this technique thus is limited to traits which are found within the same plant species. Alternatively, exposure to mutagenic agents can also introduce traits which may produce changes in the composition of a plant seed oil. However, it is important to note that Fatty Acid Synthesis (FAS) occurs in most tissues of the plant including leaf (chloroplasts) and seed tissue (proplastids). Thus, although a mutagenesis approach can sometimes result in a desired modification of the composition of a plant seed oil, it is difficult to effect a change which will not alter FAS in other tissues of the plant.
A wide range of novel vegetable oils compositions and/or improved means to obtain or manipulate fatty acid compositions, from biosynthetic or natural plant sources, are needed. Plant breeding, even with mutagenesis, cannot sufficiently meet this need and provide for the introduction of novel oil.
For example, cocoa-butter has certain desirable qualities (mouthfeel, sharp melting point, etc.) which are a function of its triglyceride composition. Cocoa-butter contains approximately 24.4% palmitate (16:0), 34.5% stearate (18:0), 39.1% oleate (18:1) and 2% linoleate (18:2). Thus, in cocoa butter, palmitate-oleate-stearate (POS) comprises almost 46% of triglyceride composition, with stearate-oleate-stearate (SOS) and palmitate-oleate-palmitate (POP) comprising the major portion of the balance at 33% and 16%, respectively, of the triglyceride composition. Other novel oils compositions of interest might include trierucin (three erucic) or a triglyceride with medium chain fatty acids in each position of the triglyceride molecule.
Plant seed oils contain fatty acids acylated at the sn-1, sn-2, and sn-3 positions of a glycerol backbone, referred to as a triacylglycerol (TAG). The structure of the TAG, as far as positional specificity of fatty acids, is determined by the specificity of enzymes involved in acylating the fatty acyl CoA substrates to the glycerol backbone. For example, there is a tendency for such enzymes from many temperate and tropical crop species to allow either a saturated or an unsaturated fatty acid at the sn-1 or the sn-3 position, but only an unsaturated fatty acid at the sn-2 in the seed TAGs. In some species such as cocoa, TAG compositions suggest that this tendency is carried further in that there is an apparent preference for acylation of the sn-3 position with a saturated fatty acid, if the sn-1 position is esterified to a saturated fatty acid. Thus, there is a higher percentage of structured TAG of the form Sat-Un-Sat (where Sat=saturated fatty acid and Un=unsaturated fatty acid).
Of particular interest are triglyceride molecules in which stearate is esterified at the sn-1 and sn-3 positions of a triglyceride molecule with unsaturates in the sn-2 position particularly oleate. Vegetable oils rich in such SOS (Stearate-Oleate-Stearate) molecules share certain desirable qualities with cocoa butter yet have a degree of additional hardness when blended with other structured lipids. SOSxe2x80x94containing vegetable oils are currently extracted from relatively expensive oilseeds from certain trees grown in tropical areas such as Sal, Shea, and Illipe trees from India, Africa, and Indonesia respectively. Cheaper and more conveniently grown sources for SOS -type vegetable oils are desirable.
In addition, vegetable oils rich in stearate fatty acid content tend to be solid at room temperature. Such vegetable fats can be used directly in shortenings, margarine and other food xe2x80x9cspreadxe2x80x9d products, obviating the need for chemical hydrogenation. Hydrogenation is a process whereby molecular hydrogen is reacted with the unsaturated fatty acid triglyceride until the desired degree of solidity is obtained. The solidity is commonly determined by the solid fat index (SFI, Official and Tentative Methods, American Oil Chemists"" Society, Cd 10-57(93), Champaign, Ill.). Values are determined by dilatometry (expansion in volume) over a defined temperature range of 50xc2x0, 70xc2x0 80xc2x0, 92xc2x0 and 100xc2x0 or 104xc2x0 F. The hydrogenation process converts unsaturated fatty acids to partially or fully saturated fatty acids, and increases the heat and oxidative stability of the product. The iodine value (IV) measures the degree of unsaturation of a fat. Lower values indicate greater saturation. The oxidative stability may be measured by an oil stability index (Official and Tentative Methods, American Oil Chemists"" Society, Cd 1b-87, Champaign, Ill.) and active oxygen method (AOM, Official and Tentative Methods, American Oil Chemists"" Society, Cd 12h-92, Champaign, Ill.). The cost and any other factors associated with chemical hydrogenation, such as the production of trans fatty acids, can be avoided if the vegetable oil is engineered to be stearate rich in the plant seed.
Moreover, some plant tissues use 18 carbon fatty acids as precursors to make other compounds. These include saturated long chain fatty acids longer than 18 carbons in length. Since very little stearate typically accumulates in soybean plants, it may be necessary to increase stearate accumulation if one wants to increase production of compounds which depend upon supply of stearate fatty acids for synthesis.
The fatty acid composition of soybean oil described above is often considered less than optimal in terms of oil functionality. While the limitations of the fatty acid composition may be partly overcome by chemical hydrogenation, the trans fatty acids produced as a result of the hydrogenation process are Sat-Un-Satpected of having unfavorable health effects (Mensink, et al. (1990) N. Eng. J. Med. 323:439-445).
Through the efforts of traditional plant breeding techniques, the fatty acid composition of soybeans has been improved. For example, using mutagenesis, plant breeders have been able to increase the amount of stearate (C18:0) produced in the soybean oil. In such high stearate lines, designated as A6 (ATCC Accession No. 97392, Hammond and Fehr, (1983) Crop Science 23:192), stearate levels of up to about 25 weight percent of the total fatty acid composition have been achieved. Such high stearate containing lines have been further bred with mutant soybean lines containing elevated levels of palmitate (16:0). Soybean lines containing the elevated stearate levels produced by mutagenesis demonstrate a negative correlation of increased stearate content and seed yield (Hartmann, et al. (1997) Crop Science 37:124-127). Attempts to further increase the stearate content and/or improve the seed yield of such increased stearate lines by breeding have thusfar proven unsuccessful.
List, et al. ((1996) J. Am. Oil. Chem. Soc. 73:729-732) describes the use of genetically modified soybean oils in margarine formulations. High stearate oil from soybean variety A6 was found to have an insufficient solid fat index at 24.7xc2x0 C. and higher temperatures to make margarine. The soybean oil was blended with cottonseed or soybean hardstocks to afford mixtures with sufficient solids content for formulation into margarine.
While soybean based products are a major food source, improvements to the nutritional and commercial quality of this product could add further value to soybean based products. Alteration of the soybean oil content and composition could result in products of higher nutritional content and greater stability. The need for industrial hydrogenation of polyunsaturated oil for food applications could be reduced by the preparation of soybean oil with increased concentrations of stearate.
Hammond and Fehr, (1983) Crop Science 23:192 provides the registration of the A6 soybean line with elevated stearate levels. Lundeen, et al. (1987) Crop Science 27:1102-1105 and Hartmann, et al. (1997) Crop Science 37:124-127 describe the negative association of seed yield with the elevated stearate levels produced using the fasa allele. Fehr and Hammond, U.S. Pat. No. 5,714,668 provides a soybean with an elevated level of stearic acid ranging between 20% and 32% with a low level of liolenic acid (less than 3.0%). Fehr and Hammond, U.S. Pat. No. 5,557,037 provides a soybean plant capable of producing an oil with a palmitic acid content of about 14% to about 24% and a stearic acid content of from about 20% to about 30%.
The present invention is directed to methods for producing soybean oil having high levels of stearate (C18:0). The method of producing a soybean oil containing increased levels of stearate comprises expression of an acyl-ACP thioesterase capable of producing C18:0 in the seed tissue of the soybean. In particular, the acyl-ACP thioesterase has substantial activity toward 18:0 acyl-ACP substrates, and preferably has little or no activity towards 16:0 acyl-ACP substrates.
The method generally comprises growing a soybean plant containing a construct comprising as operably linked components in the 5xe2x80x2 to 3xe2x80x2 direction of transcription, a transcription initiation region functional in a seed tissue and a DNA encoding an acyl-ACP thioesterase with substantial activity towards 18:0 acyl-ACP substrates and a transcription termination sequence.
The stearate content of the soybean oil preferably comprises greater than 20%, more preferably greater than about 33% of the fatty acid moieties in the oil. The oil of the present invention may be used as a blending source to make a blended oil product, or it may also be used in the preparation of food.
In another embodiment of the present invention, a soybean oil having an increased saturated fatty acid composition is provided. Soybean oils with saturated fatty acid compositions of greater than 50 weight percent are exemplified herein.
In yet another embodiment of the instant invention, the novel soybean oil, comprising the increased total saturated fatty acid compositions, provides a novel source of structured TAG of the Sat-Un-Sat (saturated-unsaturated-saturated) form.
The novel soybean oil as well as the soybean seed containing the novel oil finds use in many applications.